Abstract The CDC lists MDR Neisseria gonorrhoeae (Ng) as one of the three most urgent antibiotic resistance threats in the United States. A Gram-negative fastidious organism, Ng causes gonorrhea, the second-most prevalent sexually transmitted bacterial infection (STI) with >800,000 estimated cases in the United States annually. Left untreated, gonorrhea can cause pelvic inflammatory disease in women, leading to fallopian tube scarring and infertility or may disseminate, causing joint and skin manifestations. Once easily treatable, Ng has evolved resistance to nearly every antibiotic used to treat it, leaving a combination of azithromycin (AZM) and ceftriaxone (CTX) as the only currently available treatment option. Importantly, a cluster of cases was recently reported in Hawaii that was resistant to both AZM and CTX, highlighting the critical need for new therapeutics targeting antibiotic-resistant Ng infections. Bacterial translation is plagued by transcription errors, mRNA damage, and translational frameshifting events that result in non-stop ribosome complexes, preventing release of protein products and inhibiting further translation. Recovery of non-stop complexes is a crucial bacterial process mediated by trans-translation, ArfA or ArfB acting on a region of the bacterial ribosome highly conserved across all sequenced bacterial genomes. In preliminary studies, we demonstrated that oxadiazole-based compounds inhibit non-stop ribosome rescue, acting as potent antimicrobials against a range of pathogens, including Ng, with MIC90 values against Ng ranging from 0.8-1.6 M (0.25-0.54 g/mL). These compounds exhibit minimal toxicity towards mammalian cells, excellent pharmacokinetics and in vivo antibiotic activity in a F. tularensis mouse model. The objective of this proposal is to optimize the lead oxadiazole inhibitor of non-stop ribosome rescue into a novel class of broad-spectrum therapeutic agents for use against Neisseria gonorrhoeae and to advance this compound towards Investigational New Drug (IND)-enabling GLP toxicology and safety pharmacology studies and pre-IND submission. We will accomplish seven specific aims to achieve this objective: In Aim 1 we will optimize the lead series through SAR-driven analog generation. In Aim 2 we will prioritize lead series analogs through in vitro biology and ADME evaluations. In Aim 3 we will confirm and further explore mechanism of action and specificity of oxadiazole inhibitors of non-stop ribosome rescue in Ng. In Aim 4 we will select a preclinical candidate and backup based on in vivo properties. In Aim 5 we will conduct IND-enabling pharmacokinetic, toxicology and safety pharmacology studies. In Aim 6 we will perform CMC studies, targeting 1 non-GMP patch of drug substance at a GMP manufacturer. In Aim 7 we will request a pre-IND meeting with the FDA.